Field
This invention relates generally to the field of radio-assays and specifically to a reagent and method for detecting the presence of hepatitis in a sample of human blood serum.
Very broadly, the term radioassay refers to the use of radioactively labelled (tagged) substances to determine the concentration or presence of a substance in a fluid. Typically, radioassays are used to determine very small amounts of a given substance. The term radioimmunoassay (RIA) refers to the use of tagged substances in an antibodyantigen reaction to determine the presence or concentration of either substance. A typical RIA for a given substance (against which there are available antibodies) is based on the observation that a known amount of that substance (labelled) will tend to compete with an unknown amount of that substance (unlabelled) for a limited number of complexing sites on the antibodies. Hence, by counting the radioactivity of the products of such reaction, it is possible to determine an unknown concentration.
An essential step in RIA is the separation of immunochemically complexed products from the incubation solution. Ideally, the separation should be complete, relatively simple, and quick. It has been recognized that separation is greatly facilitated by using antibodies or antigenic substances which have been immobilized by attachment to essentially water-insoluble carrier materials. When such carrier materials are used in RIA, the technique is commonly referred to as solid phase RIA or SPRIA. The present disclosure is concerned with a SPRIA useful in detecting the presence of hepatitis associated antigen (HAA) in human blood serum.
Prior Art
It is well known that antibodies and antigenic substances can be immobilized by chemical attachment to a wide variety of carriers. Such attachments to organic carrier are described, for example, in U.S. Pat. No. 3,555,143, to Axen et al. Methods of attaching antibodies via intermediate silane coupling agents to inorganic materials are described in U.S. Pat. No. 3,652,761 to Weetall. More recently, in U.S. Pat. application Ser. No. 447,252, filed Mar. 1, 1974 in the names of W. Vann et al., entitled "Solid Phase Radioimmunoassay", and assigned to the present assignee, there are disclosed in detail SPRIA methods for determining concentration of digoxin, insulin, and estriol. Antibodies which can complex with those substances are coupled via silanes to porous glass particles having particle sizes ranging in the mircron and sub-micron range.
Although the technology for employing SPRIA techniques to test for HAA (hepatitis) has been available for some time, surprisingly, the commercialization of that technology has been rather limited. See, for example, the relatively recent summary entitled "Radioassay Test Kits and Components", Laboratory Management, Sept. 1974, pp. 29- 42. See also, recently issued U.S. Pat. No. 3,867,517 to Chung-Mei Ling which discloses a solid phase technique for detecting HAA. That disclosure employs a so-called "Sandwich" technique for detecting HAA in which HAA is sandwiched between an immobilized anti-HAA antibody and a tagged anti-HAA antibody. In one embodiment, the untagged antibodies are immobilized by attachment to the inner surfaces of a test tube.
We have found that by carefully combining certain features of the above disclosures with our own discovery, a very sensitive and relatively quick test for HAA is possible. Detailed descriptions of our reagents and methods are given below.